Attenuated Vaccine Against Fish Pathogen Francisella Sp.

ABSTRACT

An attenuated bacteria has been made by an insertion mutation in the iglC gene of  Francisella asiatica , by allelic exchange. The attenuated strain proved to be an effective vaccine by providing protection against an infection of  F. asiatica  in tilapia, and is believed would at least partially immunize fish from other species of  Francisella . The vaccine of the attenuated  Francisella asiatica  ΔiglC mutant can also serve as vectors to present antigens from other pathogens to the fish, thereby serving as vaccines against other pathogens as well. In addition, a highly sensitive and specific assay that can be used for the specific identification of  F. asiatica  in fish has been developed.

The benefit of the Sep. 14, 2009 filing date of U.S. provisionalapplication Ser. No. 61/242,111 is claimed under 35 U.S.C. §119(e).

This invention was made with United States government support undergrant no. USDA/CSREES 2009-36100-06293 awarded by the United StatesDepartment of Agriculture. The government has certain rights in thisinvention.

This invention pertains to fish vaccines, particularly to certainattenuated bacterial vaccines against fish pathogens of the genusFrancisella, especially Francisella asiatica.

Immune responses to live vaccines are generally of greater magnitude andof longer duration than those produced by killed or subunit vaccines,particularly against facultative intracellular bacteria. A single doseof a live-attenuated vaccine can provide better protection against laterinfection by the wild type organism, because the attenuated organismpersists and metabolizes within the host, and in some cases mayreplicate in the host for a time. Live viruses will better triggercell-mediated immune responses, which play a crucial role in controllinginfection due to intracellular pathogens. Injected vaccines areimpractical for most large commercial fish cultures due to size ofenclosure, number of individual animals, and low value per individualfish. Immersion or oral delivery of killed viruses in fish has yieldedinconsistent results. The invasion, persistence, and replication of liveattenuated vaccines result in a more effective and inexpensive vaccine.See, U.S. Pat. No. 6,010,705. Currently there are no live attenuatedvaccines for fish for the facultative intracellular pathogenFrancisella.

Members of the genus Francisella are small pleomorphic, Gram-negativebacteria, belonging to the gamma group of the class Proteobacteria. ManyFrancisella spp. are facultative intracellular pathogens of macrophagesand other various cell types of humans, rabbits, rodents, non-humanprimates, amoebas, arthropods and fish. Members of the genus Francisellaare fastidious facultative bacteria that have been found to infect agreat variety of animals (including humans), but very little is knownregarding the virulence mechanisms and virulence factors of this genus(Barker and Klose 2007; Keim et al. 2007). The different subspecies ofF. tularensis have been found to exist within macrophages in differentvertebrate hosts, arthropods, and in amoebae (Keim and Wagner 2007; Abdet al. 2003; Vonkavaara et al. 2008). Several genes provide the pathogenwith properties for survival in the extracellular compartment and alsofor survival and multiplication inside of potent phagocytes likeneutrophils and macrophages (Baron and Nano 1998; Allen 2003; Nano etal. 2004).

Francisella asiatica and Francisella noatunensis are two recentlydescribed members of the genus that cause piscine francisellosis in awide variety of fish species (Mikalsen et al. 2009). Francisellaasiatica was also previously called Francisella noatunensis subsp.orientalis. Francisella spp. are emergent bacterial pathogens that causeacute to chronic disease in warm and cold water cultured and wild fishspecies. During the past 5 years the bacteria have been implicated asthe cause of mortalities in tilapia and other important warm and coldwater species cultured in the USA, Taiwan, Costa Rica, Latin America,Hawaii, Norway, Chile, and Japan (three line grunt (Parapristipomatrilineatum), Kamaishi et al. 2005; tilapia (Oreochromis sp.), Hsieh etal. 2006; hybrid striped bass (Morone chrysops x M. saxatilis), Ostlandet al. 2006; Atlantic salmon (Salmo salar), Birckbeck et al. 2007;tilapia, Mauel et al. 2007; Atlantic cod (Gadus morhua L.), Mikalsen etal. 2007; cod, Ottem et al. 2007; and tilapia, Soto et al. 2009a). F.asiatica has been identified from the tilapia and three line grunt(Mikalsen et al. 2009). Infected fish show non-specific clinical signssuch as erratic swimming, anorexia, anemia, exophthalmia and highmortality. Upon gross and microscopic examination, several internalorgans (mainly spleen and kidney) are enlarged and contain widespreadmultifocal white nodules. Histological examination reveals the presenceof multifocal granulomatous lesions, with the presence of numeroussmall, pleomorphic, cocco-bacilli (Soto et al. 2009a).

Francisella tularensis is the most important species belonging to thisgenus (Dennis et al. 2001; Sjostedt 2007). Besides being an importantanimal pathogen, F. tularensis is a zoonotic agent which has receivedconsiderable study as a potential bioterrorism agent. The organism has ahigh infectivity rate and multiple infectious routes (Keim et al. 2007;Nano and Shmerck 2007). The genetic basis of F. tularensis virulence isstill poorly understood although several virulence determinants havebeen identified (Golovliov et al. 2003; Nano et al. 2004; Barker andKlose 2007). Previous studies have described the intracellularlocalization, survival, replication and escape of F. tularensissubspecies, in adherent mouse peritoneal cells, a mouse macrophage-likecell line J774A.1, and a human macrophage cell line THP-1. (Baron andNano 1998; Golovliov et al 2003; de Bruin et al. 2007). Some of the mostinteresting genes identified in F. tularensis are the genes of theintracellular growth locus (iglA, iglB, iglC, and iglD) present as partof a 30 Kb pathogenicity island (Nano et al. (2004; Barker and Klose2007). Igl proteins appear to be essential for the ability of F.tularensis to survive inside the macrophages and cause disease(Golovliov et al. 1997; Nano et al. 2004; Lai et al. 2004; Lauriano etal. 2004; Santic et al. 2005; Brotcke et al. 2006; de Bruin et al.2007). Recent data have shown that IglA and IglB are part of a novelFrancisella Pathogenicity Island (FPI) encoding Type Six SecretionSystem (T6SS) (Nano and Schmerk, 2007; Ludu et al. 2008-b). Mutations ofthese four Igl genes in F. tularensis have shown decreased pathogenicityof the bacterium both in vivo and in vitro in mammalian and insecttissues and cell lines (Lauriano et al. 2003; Nano et al. 2004; de Bruinet al. 2007; Vonkavaara et al. 2008).

PCR and sequence comparison of the 16S rRNA have made it possible toplace the fish Francisella asiatica at 97% similarity to F. tularensis,98% similarity to F. philomiragia, and 99% to other strains isolatedfrom fish species (Kamaishi et al. 2005; Hsieh et al. 2006; Ostland etal. 2006; Mauel et al. 2007; Mikalsen et al. 2007; Ottem et al. 2007;Soto et al. 2009). Francisella philomiragia subsp. noatunensis (nowcalled F. noatunensis) and F. piscicida were recovered from moribundfarmed Atlantic cod in Norway (Mikalsen et al. 2007; Ottem et al. 2007)displaying chronic granulomatous disease. Strains from cod in Norwayhave been characterized by phenotypic and molecular taxonomic methods asclosely related members of F. philomiragia subsp. philomiragia (Mikalsenet al. 2007; Ottem et al. 2007).

Tilapia is one of the most important cultured species in the world.Tilapia is a generic term used to designate a commercially importantfood group of fish that belong to the family Cichlidae. There are threeknown genera of tilapia, Tilapia, Sarotherodon, and Oreochromis. Inaddition, many hybrids of tilapia are known (Chapman 1992). Worldwidetilapia aquaculture production, mainly Nile tilapia (Oreochromisniloticus), has been increasing in exponential proportions during thelast decade, to greater than 2.5 million tons in 2005. One suchcommercial hybrid is red tilapia (Oreochromis mossambicus x O.niloticus). The main producing countries are China, Ecuador, Egypt,Israel, Indonesia, Singapore, Philippines and Thailand, but Mexico,Costa Rica, Honduras and other Latin-American countries have more thandoubled their production in the past five years. The United States ofAmerica is the country that imports the highest amount of tilapia,receiving more than 80% of worldwide tilapia exports (Josupeit 2008). Asthe tilapia aquaculture industry expands, tilapia farms are oftenchallenged with disease outbreaks, which in several cases have causedsevere economic losses, due to high mortality events, decreased weightgain, antibiotic and treatment expenses, etc.

Real-time PCR is a well known molecular technique that is currently usedin many laboratories for diagnosis of microbial pathogens including thefastidious bacteria Mycobacterium spp., Bacillus anthracis, F.tularensis, and organisms that are non-culturable on cell free media,the Rickettsia spp. and viruses (Bode et al. 2004; Kocagoz et al. 2005;Kidd et al. 2008; Tomaso et al. 2007; Abril et al. 2008; Takahashi etal. 2007). In recent years, fish disease diagnosticians have used thistechnique to identify and quantify bacterial, viral and parasitic fishpathogens such as: Aeromonas salmonicida, Flavobacterium columnare,Renibacterium salmoninarum, Henneguya ictaluri, Largemouth bass virus,and recently Francisella piscicida (now named F. noatunensis) inNorwegian cod (Balcazar et al. 2007; Getchell et al. 2007; Panangala etal. 2007; Suzuki & Sakai 2007; Griffin et al. 2008; Ottem et al. 2008).The high sensitivity, high specificity, and short turnaround time forresults make this technique an attractive replacement method forconventional diagnostic techniques (Espy et al. 2006).

We have identified and isolated a fish pathogen, Francisella asiaticaLADL07-285A, a clinical isolate from diseased tilapia Oreochromisniloticu. We then identified in this isolate homologue genes of the F.tularensis intracellular growth locus (iglA, iglB, iglC, and iglD). Wemade an insertion mutation in the iglC gene of LADL 07-285A, Francisellaasiatica, by allelic exchange using an insertion of a selective marker,and found that the iglC mutant was attenuated using intraperitoneal andimmersion challenges in tilapia. Laboratory challenge methods forinducing francisellosis in tilapia were evaluated by intraperitonealinjection and immersion with serial dilutions of Francisella sp. LADL07-285A. The lethal dose 50 value, 40 days post-challenge, was 10^(−5.3)(˜1.2×10³ CFU/fish) by intraperitoneal injection and was 10⁻⁴ (2.3×10⁷CFU/ml of tank water) by immersion. The mutants retained their invasivequalities, yet were cleared by the host after a short time. We haveshown that the attenuated strain provided protection against aninfection of F. asiatica in tilapia, and believe that it would be aneffective vaccine against a Francisella asiatica infection in otherfish, for example, striped bass, hybrid striped bass, and three linegrunt. In addition, the attenuated bacteria could be used to at leastpartially immunize fish from other species of Francisella. We alsodiscovered that the attenuated vaccine may be used not only to vaccinatefish against Francisella, but also to serve as a vector to presentantigens from other pathogens to the fish immune system, thereforeserving as vaccines against other known pathogens, for exampleSalmonella, of fish as well. We have also developed a highly sensitiveand specific assay that can be used for the specific identification ofF. asiatica in fish.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 illustrates the results of PCR amplification of iglC from wildtype and isogenic mutant Francisella asiatica LADL 07-285A. Lanes 1 and5 represent the standard 1 kb Ladder (STD). Lanes 2 and 6 represent thePCR amplification of the iglC gene from Francisella sp. LADL 07-285A(WT) using primer sets F46-F47 (SEQ ID NOS: 3 and 4) and F31-F38 (SEQ IDNOS: 12 and 154), respectively. Lanes 3 and 7 represent the PCRamplification of isogenic mutant strain Francisella sp. LADL 07-285AΔiglC (ΔiglC) using primer sets F46-F47 and F31-F38, respectively. Lanes4 and 8 represent the control lanes (C) using water.

FIG. 2 illustrates the mortality rate over time of tilapia challengedwith Francisella asiatica LADL 07-285A by intraperitoneal (IP) injectionusing various concentrations of the pathogen (10 fish were infected pertreatment).

FIG. 3 illustrates the mortality rate over time of tilapia challengedwith Francisella asiatica LADL 07-285A by immersion challenge usingvarious concentrations of the pathogen (10 fish were infected pertreatment).

FIG. 4 illustrates the percent mortality after 30 days post challenge oftilapia (Oreochromis sp.) challenged by immersion challenge (IC) andintraperitoneal challenge (IP) with either Francisella asiatica LADL07-285 A wild type or with F. asiatica LADL 07-285 A ΔiglC: Lane A, IPchallenge of Wild type (˜3×10⁸ CFU/fish); Lane B, IP challenge of ΔiglC(˜3×10⁸ CFU/fish); Lane C, IP challenge of Wild type (˜1.5×10⁸CFU/fish); Lane D, IP challenge of ΔiglC (˜1.5×10⁸ CFU/fish); Lane E, ICchallenge of Wild type (˜3.7×10⁷ CFU/ml); Lane F, IC challenge of ΔiglC(˜3.7×10⁷ CFU/ml); Lane G, IC challenge of Wild type (˜1.8×10⁷ CFU/ml);and Lane H, IC challenge of ΔiglC. Wild type (˜1.8×10⁷ CFU/ml).

FIG. 5A is a histological photomicrograph of un-infected tilapia spleen40 days post infection stained with H&E, showing a normal splenicparenchyma and stroma.

FIG. 5B is a histological photomicrograph of a severely infected tilapiaspleen 40 days post infection with Francisella asiatica stained withH&E, showing widespread multifocal granulomatous lesions with mixedinflammatory infiltrates (23 CFU/ml immersion exposure).

FIG. 6 illustrates the mean percent survival of tilapia vaccinated withdifferent treatments of Francisella asiatica ΔiglC mutant by immersion,or mock vaccinated with PBS (Controls) and challenged 4 weeks later withWT F. asiatica. Fish were vaccinated with: A. 10⁷ CFU/ml of the F.asiatica ΔiglC mutant for 180 min. B. 10⁷ CFU/ml of the F. asiaticaΔiglC mutant for 30 min. C. 10³ CFU/ml of the F. asiatica ΔiglC mutantfor 180 min. D. 10³ CFU/ml of the F. asiatica ΔiglC mutant for 30 min.E. PBS for 180 min. Four weeks post-immunization fish were challengedwith 10⁸ CFU/ml of WT F. asiatica for 180 min. Mean percent survival wascalculated 30 days post-challenge with WT. Each bar represents the meanpercent survival±standard error of three tanks (15 fish/tank). *Denotessignificant differences, P<0.05 with respect to the control group by aStudent's t-test.

FIG. 7 illustrates the serum anti-F. asiatica antibody response inactively immunized tilapia fingerlings. Fish were vaccinated with: A.10⁷ CFU/ml of the F. asiatica ΔiglC mutant for 180 min. B. 10⁷ CFU/ml ofthe F. asiatica ΔiglC mutant for 30 min. C. 10³ CFU/ml of the F.asiatica ΔiglC mutant for 180 min. D. 10³ CFU/ml of the F. asiaticaΔiglC mutant for 30 min. E. PBS for 180 min. Four weekspost-immunization fish were challenged with 10⁸ CFU/ml of WT F. asiaticafor 180 min. Each point represents the mean OD value±standard error of 5fish samples (serum). *Denotes significant differences, P<0.05 withrespect to the control group by a Student's t-test.

FIG. 8 illustrates the mucus anti-F. asiatica antibody response inactively immunized tilapia fingerlings. Fish were vaccinated with: A.10⁷ CFU/ml of the F. asiatica ΔiglC mutant for 180 min. B. 10⁷ CFU/ml ofthe F. asiatica ΔiglC mutant for 30 min. C. 10³ CFU/ml of the F.asiatica ΔiglC mutant for 180 min. D. 10³ CFU/ml of the F. asiaticaΔiglC mutant for 30 min. E. PBS for 180 min. Four weekspost-immunization fish were challenged with 10⁸ CFU/ml of WT F. asiaticafor 180 min. Each point represents the mean OD value±standard error of 5fish samples (mucus). * Denotes significant differences, P<0.05 withrespect to the control group by a Student's t-test.

FIG. 9 illustrates the enhanced antibody-dependent phagocytosis of F.asiatica by tilapia head kidney derived macrophages (HKDM). F. asiaticawas opsonized with heat-inactivated immunized (HIIS) or heat-inactivatednormal (HINS) sera obtained from adult tilapia. Results are shown asmean Log₁₀ CFU/ml of F. asiatica uptake in HKDM at 0, 24, and 48 h timepoint. The error bars represent standard error of triplicate samples andthe results shown are representative of three independent experiments.Different letters denote significant differences between treatments,P<0.05.

FIG. 10 illustrates the adoptive transfer of heat-inactivated normalserum (HINS), heat-inactivated immunized serum (HIIS) or PBS to naïvetilapia fingerlings. Mean percent mortality for each treatment wascalculated 21 days post-challenge with wild type. Each bar representsthe mean percent mortality±standard error of three tanks (20 fish/tank).*Denotes significant differences, P<0.05 with respect to the controlgroup (PBS) by a Student's t-test.

We examined the presence of homologues to the Igl virulence genes innon-tularensis fish pathogenic Francisella asiatica, and discovered auseful method for allelic exchange using PCR products to mutate F.asiatica. We then created an attenuated iglC mutant which was testedusing both intraperitoneal and immersion challenges in tilapia(Oreochromis sp.). We also examined intraperitoneal and immersioninfectivity trials, to induce francisellosis in tilapia (Oreochromissp.), and report the dose required to cause mortality in 50% of the fish(LD₅₀) of this important emergent fish pathogen. We have created anattenuated bacterial mutant which can be used as a vaccine to protectfish from infection with F. asiatica. The F. asiatica strain utilized,LADL 07-285A, was isolated from tilapia from Costa Rica, CentralAmerica, at the Louisiana Aquatic Diagnostic Laboratory, LSU School ofVeterinary Medicine, and was confirmed by molecular analysis as F.asiatica and exhibited 99% identity with other fish pathogenicFrancisella spp. After genetic comparison, the isolates from Costa Ricawere found to belong to the same species as the earlier isolates fromJapan and Taiwan, both of the species Francisella asiatica.

In other work, we examined the interaction of Francisella asiatica wildtype and a F. asiatica ΔiglC mutant strain with fish serum and headkidney derived macrophages (HKDM) of tilapia. Both the wild type and themutant strains were shown to be resistant to killing by normal andheat-inactivated serum. The wild type F. asiatica was able to invadetilapia head kidney derived macrophages and replicate vigorously withinthem, causing apoptosis and cytopathogenicity in the macrophages 24 and36 h post infection. In contrast, the F. asiatica ΔiglC mutant was foundto be defective for survival, replication, and the ability to causecytopathogenicity in HKDM, but the ability is restored when the mutantis complemented with the iglC gene. Uptake by the HKDM was partiallymediated by complement and partially by macrophage mannose receptors, asdemonstrated by in vitro assays. See, E. Soto et al. 2010b; and U.S.Provisional Application 61/242,111.

EXAMPLE 1 Isolation of Francisella sp. Materials and Methods

Fish history: Approximately 50 tilapia, Oreochromis niloticus (L.),cultured in the province of Alajuela, Costa Rica, were received andanalyzed by the Pathology Service of the School of Veterinary Medicineof the Universidad Nacional de Costa Rica during August-October 2007.Fifteen euthanized fish were sent to the Louisiana Aquatic DiagnosticLaboratory (LADL) at Louisiana State University-School of VeterinaryMedicine (LSU-SVM), Baton Rouge, La., for further analysis.

Histological analysis: At LSU-SVM, the gill, spleen, kidney, liver,heart, brain, ovary, testis and muscle were fixed in neutral buffered10% formalin; processed by standard methods, and stained withhaematoxylin and eosin and Giemsa stain, and examined by lightmicroscopy. Unless otherwise stated, all chemicals and materials werecommercially purchased from Sigma Chemical Co., St. Louis, Mo.

Isolation, media and growth conditions for bacteria: Fish tissues(spleen, anterior kidney and liver) were aseptically collected and usedfor bacteriological analysis by streaking on different agar media.Commercially available media tested for primary recovery of bacteriafrom fish tissue smears included: trypticase soy agar (“TSA”) with 5%sheep blood, cystine heart agar (“CHA”) with rabbit blood andantibiotics, chocolate agar/improved Thayer-Martin biplate (Remel,Lenexa, Kans.), chocolate II agar (GC II agar with haemoglobin andIsovitalex), and modified Thayer-Martin agar (Becton Dickenson (BD) BBL,Sparks, Md.). Two types of agar plates used as primary isolation mediawere prepared in the media preparation laboratory at LSU-SVM: cystineheart agar supplemented with bovine haemoglobin solution (CHAH) (BectonDickenson (BD) BBL, Sparks, Md.) and Mueller-Hinton base supplementedwith 3% foetal bovine serum, 1% glucose and 0.1% cystine. Polymixin B100 units mL⁻¹ and/or ampicillin 50 μg mL⁻¹ were added to the media toselect against secondary contaminants.

Plates were incubated at 22-25° C. for 2-5 days. Colonies observed fromprimary isolation agar plates were re-plated for purity of culture underthe same conditions. Once single colonies were observed and purity ofthe isolate determined, the isolate was re-suspended in liquid medium asreported by Baker, Hollis & Thornsberry (1985) with modifications. Theliquid medium consisted of a modified Mueller-Hinton II cation adjustedbroth supplemented with 2% IsoVitaleX (BD BBL) and 0.1% glucose (MMH).Broth cultures were grown overnight at 22° C. in a shaker at 175 rpm,and bacteria were frozen at −80° C. in the broth media containing 20%glycerol for later use.

Three different isolates (obtained from three different fish) weretested at different culture temperatures; 15, 20, 22, 25, 28, 30, 32, 35and 37° C. on CHAH for a period of 7 days to find the in vitro optimalgrowth temperature of the bacteria. The isolate labeled 07-285 A wasused to make the attenuated mutant bacteria.

DNA extraction: Two isolates (07-285A and 07-285B) recovered from fishas described above were used for molecular analysis. A loop of thebacterium was suspended in 400 μL of sterile water, washed andcentrifuged at 3000×g for 5 min, and re-suspended in 200 Dulbecco'sphosphate-buffered saline (PBS; Gibco/Invitrogen, Carlsbad, Calif.). Thebacterial suspension was subjected to DNA extraction and purification asper the manufacturer's protocol using the High Pure PCR TemplatePreparation Kit (Roche). DNA was stored at 4° C. until further use.

PCR and 16S rRNA gene sequence: Two different sets of primers were usedduring the study to amplify gene sequences important in identificationof the genus Francisella. The 50 μL Francisella.-specific PCR reactionwas composed of 0.2 μM of each primer (F11, 5′-TAC CAG TTG GAA ACGACTGT-3′) (SEQ ID NO:17) and F5,5′-CCT TTT TGA GTT TCGCTC C-3′) (SEQ IDNO:18) developed by Forsman, Sandtstrom & Sjostedt (1994), 0.2 mM ofdNTPs, 2.5 mM MgCl₂, 5 U of Taq DNA polymerase (AppliedBiosystems-Roche, Foster City, Calif.), 1×PCRx Amp buffer (Invitrogen,Carlsbad, Calif.), 1×PCRx Enhancer solution (Invitrogen) andapproximately 200 ng of template DNA. Cycling conditions consisted of aninitial denaturation step of 3 min at 94° C., followed by 35 cycles of30 s at 94° C., 60 s at 60° C., and 60 s at 72° C., with a finalextension step of 5 min at 72° C. performed in a Perkin Elmer GeneAmpPCR System 2400 (PerkinElmer Life and Analytical Sciences, Inc.,Waltham, Mass.).

The 50 μL universal eubacterial 16S rRNA PCR reaction was composed of0.5 μM of each primer (F1,5′-GAG TTT GAT CCT GGC TCAG-3′ (SEQ ID NO:19)and R13,5′-AGA AAG GAG GTG ATC CAG CC-3′) (SEQ ID NO:20) (Dorsch &Stackebrant 1992), 0.2 mM of dNTPs, 2.5 U of Taq DNA polymerase, 1×buffer H (Invitrogen), and approximately 200 ng of template DNA. Cyclingconditions consisted of an initial denaturation step of 30 s at 94° C.,followed by 30 cycles of 30 s at 94° C., 60 s at 58° C., and 90 s at 72°C., with a final extension step of 7 min at 72° C. in a Perkin ElmerGeneAmp PCR System 2400. The PCR products were subjected toelectrophoresis on a 1% agarose gel and stained with SYBR® Safe DNA gelstain (Invitrogen).

Amplicons for sequencing were purified with the QiaQuick PCR Cleanup Kit(Qiagen, Valencia, Calif.) as directed by the manufacturer and weresequenced on an Applied Biosystems 3130 Genetic Analyzer using PCRprimers (F11-F5) and (F1-R13).

Experimental challenges: In order to fulfill Koch's postulates,experimental infections were performed by intraperitoneal injection (IP)and gill spraying (GS) with the Francisella asiatica Costa Rica isolateLADL07-285A. This isolate, recovered from cultured infected tilapia inCosta Rica was grown in CHAH at 25° C. for 72 h. Cells were harvested,suspended in 5 mL of MMH broth, and incubated in a shaking incubatorovernight at 22° C. to obtain a final optical density at 600 nm (OD₆₀₀)of 0.48. Enumeration of the bacteria was done by the drop plate methodwith 50 μL drops of each 10-fold dilution placed on cystine heart agarwith haemoglobin. Resulting colony forming units per mL (CFU mL⁻¹) weredetermined.

Experimental infection of naïve O. niloticus (average length ˜9.0 cm andaverage weight ˜18.9 g) was tested by the IP and GS exposure routes. Thefish were obtained from a source considered to be free of Francisellainfection (TilTech Aquafarm, Robert, La.) and were found to be negativefor francisellosis by culture of spleen and head-kidney smears and byPCR, prior to use in the study. Fish were maintained in 3 differenttanks (10 fish per tank), representing the 2 different challenge methodsand a control tank at 23-25° C. Prior to challenge, all fish wereanaesthetized with MS-222 (100 mg L⁻¹). The IP challenge fish received a0.1 mL injection of the bacterial suspension (˜10⁷ CFU/fish). The GSchallenge fish were sprayed with 0.1-0.2 mL of the bacterial suspension,and left out of the water for approximately 15 s. Control fish weretreated in a similar manner, but received 0.1 mL of sterile MMH broth.

Following each challenge exposure, the fish were placed in therespective tanks and mortality was recorded every 12 h for 10 days. Deadand moribund fish were subjected to a complete clinical, bacteriologicaland histopathological examination. The identity of isolated bacteria wasconfirmed by PCR.

EXAMPLE 2 Isolation of Francisella asiatica from Fish and ChallengeTesting

Cystine heart agar supplemented with bovine haemoglobin solution andantibiotics, the modified Thayer-Martin agar, and CHA with rabbit bloodand antibiotics were useful for the primary isolation of Francisellaasiatica from the spleen and kidneys of diseased fish. The chocolateagar/improved Thayer-Martin biplate, chocolate II agar, and the MuellerHinton base supplemented with 3% foetal bovine serum, 1% glucose and0.1% cystine were not suitable for primary isolation, althoughsub-culture could be successfully performed on these agars. The F.asiatica failed to grow on TSA agar with 5% sheep blood. The strains ofF. asiatica isolated from tilapia from Costa Rica by the LADL weredesignated as strains LADL07-285A and LADL07-285B.

Growth of Francisella asiatica was visible on CHAH, 36-48 hpost-inoculation and colonies were grey, smooth and convex. Optimalgrowth of F. asiatica occurred at 28-30° C., but growth was present from20-28° C. after four days of incubation. Growth at 22-25° C. was slowerthan at 28° C., and no growth was observed at 15° C. or at 33° C. Bylight microscopy, the morphology of the bacterium was extremelypleomorphic, non-motile and very small in size (˜0.5-1 μM wide).

The isolates recovered from the infected spleen and kidneys yielded theappropriately amplified PCR products of 1150 bp using the Francisellagenus-specific primers F11 (SEQ ID NO:17) and F5 (SEQ ID NO:18) (Datanot shown). When using the universal eubacterial 16S rRNA primers F1(SEQ ID NO:19) and R13 (SEQ ID NO:20), a 1384 bp product was amplifiedfrom LADL07-285A and LADL07-285B. The sequence for isolate F. asiaticaLADL07-285A was deposited in GenBank under the accession numberEU672884.

Intraperitoneal injection of F. asiatica LADL07-285A of ˜10⁷ CFU/fishcaused 100% mortality in naïve tilapia by 72 h post-inoculation. Tilapiaexposed to bacteria by gill immersion also exhibited high mortality(80%), but this occurred gradually over the duration of the study (10days). The clinical signs presented in the experimentally challengedfish were consistent with those found in the naturally infected cases.In the IP injection group, a more acute onset of the disease was seenand most fish died in a short period of time (<48 h post-challenge). Theclinical signs in the acutely infected fish were bloody ascites, slightswelling of the spleen and kidney, with increased number and size ofmelanomacrophage centres but no granulomas were seen. Numerous smallcocco-bacilli were present both intracellularly and extracellularly inthe tissues. Fish exposed by gill immersion presented with a moresubacute to chronic form of the disease, showing signs of anorexia anderratic swimming behavior. At necropsy, splenomegaly and renomegaly werepronounced and granulomas were numerous in both organs. Numerous intraand extracellular bacteria were observed microscopically in gills,spleen, and anterior and posterior kidney. F. asiatica was re-isolatedfrom both challenged groups by inoculating homogenates of spleen andposterior kidney on CHA supplemented with bovine haemoglobin solutionand antibiotics. The isolates were confirmed by PCR as members of thegenus Francisella.

At the completion of the experimental challenge, all control fish werealive and no bacterial infection was detected by bacteriological,histopathological or molecular analysis.

EXAMPLE 3 Materials and Methods for Development of Attenuated BacterialVaccine

Bacterial strains and growth conditions: Strains, plasmids and primersused are listed in Table 1. Francisella asiatica LADL 07-285A wasisolated from cultured tilapia (Oreochromis sp.) as described above. F.asiatica LADL 07-285A was grown in Cystine Heart Agar supplemented withbovine hemoglobin solution (BD BBL, Sparks, Md., USA) (CHAH) for 48 h at28° C. A liquid culture medium consisted of a modified Mueller-Hinton IIcation adjusted broth supplemented with 2% IsoVitaleX (BD BBL, Sparks,Md., USA) and 0.1% glucose (MMH). Broth cultures were grown overnight at25° C. in a shaker at 175 rpm, and bacteria were frozen at −80° C. inthe broth media containing 20% glycerol for later use. Polymixin B (100units/ml) and ampicillin (50 μg/ml) were added when needed to make theprimary isolation media selective to aid in recovery of the bacteriafrom fish tissues; and kanamycin (15 μg/ml) was used for recovery oftransformed bacteria following electroporation. Escherichia coli XL1Blue MRF′ was grown using Luria-Bertani broth or agar for 16-24 h at 37°C. and supplemented with kanamycin (50 μg/ml) when needed to recover theplasmid containing bacteria after electroporation.

TABLE 1 Description of strains, plasmids and primers CharacteristicsSource (if any) Bacterial Strain Francisella asiatica 07-285AIsolated from tilapia E.coli XL1 Blue MRF Plasmids pEN1 Km^(R)Ludu et al., 2008-a pBS High copy number plasmid Stratagene pBSiglCHigh copy number plasmid-wild type iglC pBSΔiglCHigh copy number plasmid with ΔiglC , Km^(R)Primers used for mutagenesis F-40 (iglC-XhoI) 5′ aatt ctcgagtgttggtgctgagcaaattc 3′ (SEQ ID NO: 1) F-41 (iglC-SpeI) 5′ aattta actagtcagcacagcatacaggcaag 3′ (SEQ ID NO: 2) F-46 (iglC) 5′tgttggtgctgagcaaattc 3′ (SEQ ID NO: 3) F-47 (iglC) 5′cagcacagcatacaggcaag 3′ (SEQ ID NO: 4) F-12 FA1451-1 5′ttttgggttgtcactcatcgt 3′ Liu et al., 2007 (SEQ ID NO: 5) F-13 FA1451-25′ cgctataaccctcttcattt 3′ (SEQ ID NO: 6) Primers used foramplification of iglABCD homologues F36iglA 5′ gggaagatcggtagatgcaa 3′(SEQ ID NO: 7) F37iglA 5′ cgagtagtgctctgatttctgg 3′ (SEQ ID NO: 8)FA22iglB 5′ gtcagaagagtaaataatggtgt 3′ Liu et al., 2007 (SEQ ID NO: 9)FA23iglB 5′ ggctctatactaatactaaaagc 3′ Liu et al., 2007 (SEQ ID NO: 10)F30iglBinternal 5′ tttagttattattcgcaccg 3′ (SEQ ID NO: 11)F31iglBinternal 5′ caggaagtttgtcaagatga 3′ (SEQ ID NO: 12) FA26iglC 5′gagtttgaaggaatgaatactacaatga 3′ (SEQ ID NO: 13) FA27iglC 5′gagccatcttcccaataaatcctt 3′ (SEQ ID NO: 14) F38iglD 5′gctggagctattgcctttctt 3′ (SEQ ID NO: 15) F39iglD 5′tgctatcctctatctttgcaggt 3′ (SEQ ID NO: 16)

Identification of F. tularensis operon iglABCD homologue in Francisellaasiatica LADL 07-285A: The complete genome sequences of F. philomiragiasubsp. philomiragia ATCC 25017 (GeneBank accession number CP000937), F.tularensis subsp. novicida U112 (GeneBank accession number CP000439),and partial genome sequences of F. piscicida strain GM2212 (GeneBankaccession number EU492905), available from the National Center forBiotechnology Information (NCBI), were used to compare the iglABCDregions. Previously published F. tularensis primers to these genes werealso compared and were used as a template to design primers to amplifyhomologous regions from the F. asiatica LADL 07-285A chromosomal DNA bypolymerase chain reaction (PCR). PCR amplicons for sequencing werepurified with the QiaQuick Minelute PCR Cleanup Kit (Qiagen, Valencia,Calif., USA) as directed by the manufacturer, and were sequenced on anApplied Biosystems 3130 Genetic Analyzer using the PCR primers in Table1.

The sequences from the F. asiatica LADL 07-285A iglABCD genes and thecorresponding amino acid sequences were compared with those stored inthe NCBI database using the BLASTN and BLASTP program, with defaultsettings.

Electroporation: Electrocompetent E. coli and F. asiatica LADL 07-285Awere prepared following Maier et al. (2004) with some modifications.Briefly, E. coli was aerobically grown until mid-logarithmic stage(OD₆₀₀ 0.7), and the cells were prepared by washing 2 times in waterfollowed by 1 wash in 10% glycerol. The electrocompetent E. coli wereelectroporated using a BioRad Gene Pulser Controller, in a 2 mmelectroporation cuvette (BTX Harvard apparatus, Holliston, Mass.). Thepulser was set at a voltage of 2.5 kV, a capacitance of 25 uF, and aresistance of 200Ω Immediately after electroporation, cells weresuspended in 1 ml of LB-broth; and incubated with shaking for 1 h at 37°C. After the 1 h incubation period, E. coli was plated on LB agar withkanamycin (50 ug/ml).

Francisella asiatica LADL 07-285A was grown aerobically untillate-logarithmic stage (OD₆₀₀ 0.6), and the cells were prepared by using0.5 M sucrose. The electrocompetent F. asiatica were electroporatedusing the Gene Pulser in a 2 mm cuvette. The pulser was set at a voltageof 2.5 kV, a capacitance of 25 uF, and a resistance of 600Ω. Immediatelyafter electroporation, cells were suspended in 1 ml of MMH-broth, andincubated with shaking for 4 h at 28° C. After the 4 h incubationperiod, F. asiatica was plated on CHAH with Kanamycin (15 ug/ml).

Mutant and plasmid construction: A fragment of approximately 850 basepairs corresponding to a portion of the iglB and iglC genes from F.asiatica LADL-07-285A was PCR amplified using primers F-40 (SEQ IDNO: 1) and F-41 (SEQ ID NO: 2) (Table 1), which contain XhoI and SpeIsites, respectively. All enzymes used during the study were supplied byNew England Biolabs, Inc. (Ipswich, Mass.), and were used under theconditions recommended by the manufacturer. The PCR product was cleavedwith these two endonucleases and ligated into the high copy numberplasmid pBluescript SK (pBS), resulting in plasmid pBS-iglC. The plasmidwas electroporated into E. coli, amplified, and then purified from thebacterium using the QIAprep Spin Miniprep Kit (Qiagen, Valencia, Calif.,USA) following the manufacturers protocol.

Plasmid pEN1, constructed and donated by Ludu et al. (2008-a), containsa Tn903 Kanamycin cassette linked to Francisella novicida promoterderived from the region upstream of gene FTN_(—)1451 (Km-P) (Gallagheret al. 2007). Purified pEN1 plasmid was digested with PstI to releasethe Km-P cassette. Other known selection markers could be used insteadof Kanamycin, for example, other antibiotics, color-expressing markers(e.g., green fluorescent protein (GFP) or M-cherry), and heat selectionmarkers.

For the construction of pBS-ΔiglC, plasmid pBS-iglC was digested withPstI endonuclease, which cuts once in the iglC gene. The 1100 bp KmPcassette was ligated into the unique PstI site in pBS-iglC, resulting inpBS-ΔiglC. The resulting insertion was verified by sequencing.

EXAMPLE 4 Identification of IglABCD Operon

The deduced amino acid products of the Francisella asiatica LADL 07-285AiglA gene have 95, 92 and 88% similarities to the intracellular growthlocus protein A of F. philomiragia subsp. philomiragia, F. piscicida,and F. tularensis subspecies respectively. The amino acid sequences ofthe F. asiatica LADL 07-285A proteins IglB, IglC and IglD showedidentity of 97, 95 and 92% (IglB), 93, 90 and 89% (IglC) and 94, 92 and80% (IglD) respectively to the intracellular growth locus proteins foundin F. philomiragia, F. piscicida, and F. tularensis species,respectively. The G+C content found in the iglABCD operon from F.asiatica LADL 07-285A (GeneBank accession number FJ386388) was 31%.Overall DNA comparison between F. asiatica LADL 07-285A, F. philomiragiasubsp. philomiragia and F. tularensis subsp. novicida U112 iglABCDoperon, showed that the F. asiatica fish pathogen shares 94% identity toF. philomiragia and 83% identity with F. tularensis subsp. novicida. TheiglABCD operon of the three members of the genus Francisella were in thesame orientation and arrangement.

EXAMPLE 5 Generation of a Francisella asiatica LADL 07-285A iglC Mutant

An insertion mutation made in the iglC gene of F. asiatica LADL 07-285Aby allelic exchange using Km-P was found to have approximately 400 basepairs of flanking sequences on either side of the insertion site.Insertion of Km-P was confirmed by PCR using 2 different set of primersand DNA sequencing. Primer sets F46-F47 (SEQ ID NOS.: 3 and 4) andF31-F38 (SEQ ID NOS: 12 and 15) were used to verify the insertion andposition of the 1100 bp Km-P cassette in iglC (FIG. 1). Primers used foramplification of the FA-1451 promoter region were also used to sequencethe inside region of the insertion, and verify the presences of thepromoter in the mutant. FIG. 1 illustrates the results of PCRamplification of iglC from wild type and isogenic mutant F. asiaticaLADL 07-285A. Lanes 1 and 5 represent the standard 1 kb Ladder. Lanes 2and 6 represent the PCR amplification of the iglC gene from F. asiaticasp. LADL 07-285A using primer sets F46-F47 (SEQ ID NOS: 3 and 4) andF31-F38 (SEQ ID NOS: 12 and 15), respectively. Lanes 3 and 7 representthe PCR amplification of isogenic mutant strain F. asiatica LADL 07-285AΔiglC using primer sets F46-F47 (SEQ ID NOS: 3 and 4) and F31-F38 (SEQID NOS: 12 and 15), respectively. Lanes 4 and 8 represent the controllanes using water.

The resulting F. asiatica LADL 7-285A ΔiglC strain had no obviousmorphological differences from the wild type strain and growthcharacteristics were identical to those of the parental strain in brothand on agar media. The insertional mutagenesis protocol followed,allowed the selection for a double recombination in the F. asiatica LADL07-285A iglC gene. Kanamycin was used as the selective antibioticresistance marker due to the natural kanamycin susceptibility of theFrancisella sp. strain used in this study (data not shown). Other knownselection markers could be used instead of Kanamycin, for example, otherantibiotics, color-expressing markers (e.g., green fluorescent protein(GFP) or M-cherry), and heat selection markers.

A sample of the novel Francisella sp. LADL 07-285A ΔiglC strain,designated Francisella asiatica ΔiglC (LSU F1) was deposited with theAmerican Type Culture Collection (ATCC), 10801 University Boulevard,Manassas, Va. 20110-2209, United States on Aug. 26, 2010, and wasassigned ATCC Accession No. ______. This deposit was made under theBudapest Treaty.

EXAMPLE 6 Tilapia LD-50 Virulence Assays

Preparation of bacterial stock culture and enumeration: Francisellaasiatica LADL 07-285A was cultivated in MMH in a shaking incubator at175 rpm overnight at 25° C. The bacteria were then pelleted and theconcentrations were adjusted to ˜2.3×10⁹ CFU/ml in Dulbecco'sphosphate-buffered saline (PBS; Gibco/Invitrogen, Carlsbad, Calif.).Ten-fold dilutions of this stock were then made in sterile saline.Actual bacterial numbers delivered by injection and by immersion weredetermined by colony counts on CHAH plates. Enumeration of the bacteriawas done by placing 50 μl drops of each 10-fold dilution on CHAH andcounting the resulting colonies after 72 h incubation at 25° C. Thedilution which produced countable colonies (10-50 per drop) was thenused to calculate the CFU/ml in the stock suspension.

Fish and systems: Naïve tilapia (average length ˜9.0 cm and averageweight ˜18.9 g) were obtained from a source believed to be free ofFrancisella infection (TilTech Aquafarm, Robert, La.), and a sub-sampleof the population was confirmed as negative for Francisella bacteria byculture on CHAH and PCR, prior to use in the study. Groups of 10 fishwere placed in 90 L tanks with filtered recirculating water flow (onetank per treatment). Water temperature was maintained in the range of23-25° C. throughout the study, and fish were fed daily with acommercial 4.7 mm pelleted fish feed (Cargill, Franklinton, La.) at 5%body weight. The fish were allowed to acclimate for at least 2 weeksprior to challenge. At challenge, all fish were anesthetized with MS-222(100 mg/l) prior to handling.

Intraperitoneal Injection (IP): From the initial bacterial concentrationin the stock suspension (2.3×10⁹ CFU/ml in PBS), 10 serial dilutions inPBS were prepared. Each fish per treatment was injected with 0.1 ml ofthe bacterial suspension. Fish in the control tank were injected with0.1 sterile PBS. Mortality was recorded daily following IP injection andthe LD₅₀ calculated for the wild type strain of F. asiatica LADL07-285A.

Immersion challenge (IC): Immersion challenge was carried out in 8different dilutions of the bacterial suspension. The IC fish wereimmersed in 10 L of static water containing 2.3×10⁸, 2.3×10⁷, 2.3×10⁶,2.3×10⁵, 2.3×10⁴, 2.3×10³, 2.3×10², 2.3×10¹ CFU of the wild type strainof F. asiatica LADL 07-285A/ml of tank water for 3 h. After 3 h, fishwere moved to a clean 90 L tank system with biofiltered recirculatingwater. Control fish were treated with sterile PBS in a similar manner.

Analysis of dead and surviving fish after challenge: Dead and survivingfish were subjected to a complete clinical and bacteriologicalexamination. A histological evaluation was performed on splenic, hepaticand renal tissue of moribund, freshly dead and surviving fish. Severityof the disease in each treatment was determined by counting the numberof granulomas in histological sections present per single 10×microscopic field from the spleen, head kidney and liver of each fish.The means of these counts were reported as relative severity in Table 2using the following scale: severe=>20, moderate=7-20, and mild=<7.Molecular analysis by PCR was performed following the above protocolusing bacterial cultures recovered from moribund and dead fish, as wellas from DNA extracted from spleen tissue of fish surviving challenge.The LD₅₀ was calculated at days 20 and 40 by the method of Reed-Muench(Anderson 1984), following both the intraperitoneal and the immersionchallenges.

In-vivo challenge with F. asiatica LADL 07-285A wild type and ΔiglC: Thewild type and ΔiglC strains were tested for virulence by both IP and ICchallenge. F. asiatica LADL07-285A wild type and ΔiglC isogenic strainswere grown on CHAH plates at 25° C. for 72 h. Cells were harvested,suspended in 1 liter of MMH broth, and incubated in a shaking incubatorovernight at 24° C. to obtain a final optical density at 600 nm (OD₆₀₀)of 0.75. Enumeration of bacteria in IP and IC challenges wasaccomplished by the same methods outlined in LD₅₀ study.

The fish were obtained from the same source, were in the same sizerange, and were fed the same way as described above for the LD₅₀ study.The challenge trials were done in 20 liter flow through tanks, however,with chlorination traps in the drain system for biosafety. Fish weremaintained at 10 fish per tank, and four tanks were used per treatmentwith one tank serving as a non-infected control. Prior to challenge allfish were anesthetized with MS-222 (100 mg/l). Intra-peritonealchallenged fish received a 0.1 ml injection of bacterial suspension(˜3×10⁸ CFU/fish, or ˜1.5×10⁸ CFU/fish). The IC fish were immersed in 8L of static water containing approximately 3.7×10⁷ CFU/ml in tank wateror 1.8×10⁷ CFU/ml of tank water for 3 h, and then the volume of thetanks was adjusted to a maximum of 20 liters with clean dechlorinatedand aerated municipal water. Control fish were treated in a similarfashion but received sterile PBS in place of the bacterial suspension.

Following each challenge exposure, mortality was recorded every 12 h for30 d. Dead fish and survivors from each challenge were subjected to acomplete clinical and bacteriological evaluation. Polymerase chainreaction was performed on DNA from bacterial cultures recovered frommoribund and dead fish to confirm the presence of wild type or ΔiglC.

Statistical analysis: Data (both original and inverse sine transformed)obtained from IC and IP challenges with the F. asiatica LADL 07-285Awild type and ΔiglC strains were compared in an analysis of variance ofa factorial arrangement of treatments with the SAS® statistical program(version 9.1.3). Where significance was found, post hoc pairwisecomparisons were conducted with t tests of least squares means.Differences were considered significant at P≦0.05.

Mortalities of tilapia challenged by IP or IC are shown in FIGS. 2 and3, respectively. Based on the cumulative mortalities found at day 20 andat day 40, the observed median lethal dose (LD₅₀) for the IP challengedtilapia infected with F. asiatica LADL 07-285A was 10^(−5.1) (˜1.8×10⁴CFU/fish), and 10⁻⁵³ (˜1.2×10⁴ CFU/fish) respectively. On the otherhand, the observed median lethal dose (LD₅₀) for the IC tilapia at day20 and at day 40, were 10^(0.52) (˜6.9×10⁷ CFU/ml), and 10⁻¹ (˜2.3×10⁷CFU/ml) respectively. The least amount of bacteria required to causemortality in the IP challenged tilapia was 23 CFU, whereas for the IC,2.3×10² CFU/ml of tank water was necessary to cause mortality (Table 2).

TABLE 2 Summary of mortalities, bacterial isolation and severity oflesions observed 40 days post challenge with Francisella asiatica LADL07-285A in LD₅₀ Virulence Assays. Bacterial Bacterial Mean value ofgranulomas in isolation isolation Survivors 10X microscopic field % fromfrom Spleen Head Challenge dose Mortality dead fish survivors PCR Spleenkidney Liver Intraperitoneal Challenge (CFU/ml of PBS) 2.3 × 10⁹ CFU/ml98.3 Pos^(a) Pos Pos Moderate^(d) Mild^(f) Mild 2.3 × 10⁸ CFU/ml 98 PosN/A^(c) Pos N/A N/A N/A 2.3 × 10⁷ CFU/ml 97.5 Pos N/A Pos N/A N/A N/A2.3 × 10⁶ CFU/ml 96.6 Pos N/A Pos N/A N/A N/A 2.3 × 10⁵ CFU/ml 86.3 PosPos Pos Severe^(d) Severe Mild 2.3 × 10⁴ CFU/ml 57.8 Pos Pos Pos MildMild Mild 2.3 × 10³ CFU/ml 27.7 Pos Pos Pos Severe Severe Mild 2.3 × 10²CFU/ml 14.2 Pos Pos Pos Severe Moderate Mild 2.3 × 10¹ CFU/ml 5.8 PosPos Pos Severe Moderate Mild 2.3 × 10⁰ CFU/ml 0 N/A Neg^(b) Pos ModerateMild Neg  2.3 × 10⁻¹ CFU/ml 0 N/A Neg Pos Mild Mild Neg  2.3 × 10⁻²CFU/ml 0 N/A Neg Neg Mild Neg Neg Control 0 N/A Neg Neg Neg Neg NegImmersion Challenge (CFU/ml of tank water) 2.3 × 10⁸ CFU/ml 78.9 Pos NegPos Severe Severe Mild 2.3 × 10⁷ CFU/ml 50 Pos Neg Pos Severe SevereMild 2.3 × 10⁶ CFU/ml 19 Pos Neg Pos Severe Severe Mild 2.3 × 10⁵ CFU/ml6.8 Pos Neg Pos Severe Moderate Mild 2.3 × 10⁴ CFU/ml 5.1 Neg Neg PosModerate Moderate Mild 2.3 × 10³ CFU/ml 4.1 N/A Neg Pos ModerateModerate Mild 2.3 × 10² CFU/ml 1.7 N/A Neg Neg Mild Mild Neg 2.3 × 10¹CFU/ml 0 N/A Neg Neg Mild Mild Neg Control 0 N/A Neg Neg Neg Neg NegLegends: ^(a)Pos = Positive ^(b)Neg = Negative ^(c)N/A = Not Applicable^(d)Severe = X > 20 ^(e)Moderate = 7 < X < 20 ^(f)Mild = X < 7 Survivingfish from both challenges were subjected to complete clinical,bacteriological, and histopathological examination at 40 days postchallenge. Selected tissues were placed in fixative at termination ofthe trial.

No obvious external clinical signs were observed in the fish.Internally, the most significant gross pathological change observed wasthe presence of widespread, multifocal white nodules dispersed in theanterior kidney, posterior kidney, and spleen, with a markedsplenomegaly and renomegaly. Histopathologically, granulomatousinflammation was present in the spleen and kidneys with large numbers ofmacrophages containing small pleomorphic coccobacilli.

F. asiatica LADL 07-285A was isolated from the spleen and kidney of deadand moribund fish from both treatments. Bacteriology, histopathologicaland molecular analysis (PCR) performed on the internal organs of fishfrom both IP and IC challenge trials are shown in Table 2.

EXAMPLE 7 In-Vivo Challenge of Francisella asiatica LADL 07-285A WildType and ΔiglC

To examine the role of the iglC gene on virulence in a fish model ofinfection, the survival rates were measured of tilapia infected with F.asiatica LADL 07-285A wild type (WT) and ΔiglC by two different routesof inoculation (IP and IC). After 48 h following IP injection of 0.1 mlof bacterial suspension (˜3×10⁸ CFU/fish, or ˜1.5×10⁸ CFU/fish), all thetilapia with the WT had died, while only one fish infected with theΔiglC died 30 days post challenge. This one dead fish recovered from thechallenge with the ΔiglC IP injection was not examined since it was inan advanced stage of decomposition. The difference in dosages did notshow significance, while the percent mortality between wild type andmutant injected fish was significantly different (P<0.0001) (FIG. 4).

The fish immersed with ˜3.7×10⁷ CFU/ml of wild type bacteria in tankwater had a survival percentage of 43.3%, and survival was 56.6% withthe groups immersed with 1.8×10⁷ CFU/ml. The dosages did not result insignificantly different mortality (P≦0.05). On the other hand, the fishchallenged with the mutant strain had a 100% survival rate whenchallenged with ˜3.7×10⁷ CFU/ml and 1.8×10⁷ CFU/ml of tank water.Percent mortality was significantly different between groups challengedwith the wild type and mutant strains (P<0.0001) (FIG. 4).

The histopathological analysis of the fish challenged with the wild typeshowed the same lesions as previously described in the LD₅₀ challenge,with increased melanomacrophage centers, widespread granulomas andgranulomatous inflammation in the spleen and head kidney. Upon gross andhistopathological analysis, the fish challenged with the ΔiglC mutant byimmersion challenge did not show any granulomatous lesions or increasednumber of melanomacrophages in the analyzed tissues. The fish challengedwith the ΔiglC mutant strain by IP injection presented higher numbers ofmelanomacrophages in the head kidney and the spleen than the controlgroup of fish injected with PBS at 30 days post challenge but nogranulomas. The control fish immersed with PBS did not display anylesions in the tissues and organs.

The iglC mutation significantly attenuated the pathogen upon in vivochallenges, and increased the survival rates of the mutant infected fishwhen compared with the wild type infected fish after both IP and ICchallenges. Two different administration routes (IP and IC) forchallenging tilapia with F. asiatica were compared and the LD₅₀ at 20and 40 days post-challenge reported. The IP challenge was chosen sinceit was an easy and quick method to accurately administer suspendedbacteria, but several problems developed when administering the bacteriaby this method, including the lack of exposure of the bacteria to innateimmune protection present in the skin, gills and other mucosa. As wasexpected, an acute onset of the disease was observed, with highmortalities and few clinical signs in the fish receiving the higherdosage. It was surprising that a low dose of bacteria, ˜0.23 CFUinjected into the peritoneum of the fingerlings, was able to causemortalities. Even more surprising was the amount and severity of lesions(granulomas), caused by a very low number of bacteria (˜1 CFU/fish), inimportant hematopoietic and osmoregulatory organs like the spleen andthe anterior kidney. Survivors of this treatment were observed withsignificant lesions in spleen, head kidney and liver, which not onlyimpair the fish's ability to osmoregulate, but also immunosuppress themby direct damage of their hematopoietic organs making them moresusceptible to other important and common tilapia diseases seen inculture facilities such as streptococcosis and columnaris disease.

The IC challenge route was chosen because it more closely resembles anatural infection. The fact that the bacteria have to come into closecontact with the innate immune system present in skin, gills,gastrointestinal mucosa, etc., more closely resembles the way thedisease progresses in nature. As expected, the amount of bacteria neededto cause mortality was higher than in the IP treatment, and the onset ofthe disease was more sub-acute to chronic, presenting anorexia, changein coloration, and pale gills. A dose of 2.3×10² CFU/ml of tank waterwas needed to cause mortality in the immersed fish, but when analyzinghistopathological lesions of the survivors, it was evident that even adose of 23 CFU/ml of tank water was able to cause significant lesions inthe spleen and head kidney (FIG. 5). The experiment was terminated 40days after exposure to the bacterium, but we suspect that the survivorsof this trial may become carriers of the pathogen as in seen in naturalinfections.

Thus, the first identification of homologous genes of the F. tularensispathogenicity island in the fish pathogenic Francisella spp. has beenreported, and an easy and reliable method for mutagenesis of thisfastidious pathogen has been developed. Data from challenge trialsindicate that mutation of iglC results in a less virulent pathogen.Based on the homology of the iglC gene in Francisella, we believe thatother fish pathogenic Francisella could be attenuated by a similarprocess to produce a ΔiglC mutant as discussed above.

EXAMPLE 8 Attenuated F. asiatica iglC Mutant Induced Protective ImmunityPart 1—Materials and Methods

Bacteria: Francisella asiatica LADL 07-285A WT was isolated fromcultured tilapia (Oreochromis sp.) as described above. The ΔiglC mutantisolate was made by homologous recombination using a PCR product, andits attenuation was demonstrated in vivo and in vitro as shown above. F.asiatica isolates were grown in cysteine heart agar supplemented withbovine hemoglobin solution (CHAH) (Becton Dickenson (BD) BBL, Sparks,Md., USA) for 48-72 h at 28° C. The liquid medium consisted ofMueller-Hinton II cation adjusted broth supplemented with 2% IsoVitaleX(BD BBL, Sparks, Md., USA) and 0.1% glucose (MMH) as described in Bakeret al. 1985. Broth cultures were grown overnight at 25° C. in a shakerat 175 rpm, and bacteria were frozen at −80° C. in the broth mediacontaining 20% glycerol for later use. Escherichia coli DH5a was grownusing Luria-Bertani broth or agar for 16-24 h at 37° C.

Preparation of sonicated F. asiatica lysate for ELISA. Approximately1×10¹² CFU of F. asiatica were harvested from 500 ml of broth culture bycentrifugation at 1,500×g for 10 min at 4° C. in a GSA rotor in anaccuspin 3R refrigerated Centrifuge (Fisher Scientific). The pellet waswashed three times with Dulbecco's phosphate-buffered saline (PBS;Gibco/Invitrogen, Carlsbad, Calif.), followed by centrifugation at1,500×g for 10 min. Following the final wash, the pellet was resuspendedin 4 ml of 20 mM Tris-Cl (pH 8.0) with a protease inhibitor cocktail(Roche Applied Sciences, Indianapolis, Ind.). The bacteria weresonicated on ice for a 30-s pulse, followed by a 30-s rest, ten timesusing a Sonic Dismembrator Model 500 (Fisher Scientific) at a power of70%. The samples were then centrifuged for 1 h at 16,000×g at 4° C. inan Eppendorf centrifuge 5415 R (Fisher Scientific). Proteinconcentration of the sonicate was determined by the Bradford proteinassay (Bio-Rad Laboratories, Hercules, Calif.).

Fish. Adult and fingerling tilapia nilotica (Oreochromis niloticus) usedduring the trial were obtained from a source with no history ofFrancisella infection (TilTech Aquafarm, Robert, La.). For verification,a sub-sample of the population was tested for, bacteria by completeclinical, bacteriological, serological and molecular analysis to ensurethat they were free of F. asiatica. Fingerlings were maintained at 15fish per tank in 40 L tanks containing 30 L of water flowing through at25° C., and fed commercial tilapia feed daily (Burris Aquaculture Feeds,Franklinton, La.) at 3% fish body weight per day. The mean weight of thefish was 6.4 g. Adults weighing an average of 346 g were acclimated fora minimum of 2 months in a flow through water system at 25° C. Fiveadult fish were maintained in a 100 L tanks containing 80 L ofwater/tank with constant oxygenation.

Immunization and challenge. Vaccination trials were conducted usingtilapia fingerlings. Four different ΔiglC mutant vaccination treatmentsand a mock immunized control treatment were evaluated. Each treatmentconsisted of eight tanks (15 fish in each tank). The first group of fishwas vaccinated by addition of 10⁷ CFU/ml of the ΔiglC mutant to 10 L ofstatic water and incubation for 180 min. The second group received adose of 10⁷ CFU/ml of the ΔiglC mutant but for 30 min. The third groupreceived a dose of 10³ CFU/ml of the ΔiglC mutant for 180 min; and thefourth group received a dose of 10³ CFU/ml of the ΔiglC mutant for 30mM. The control tanks received 100 ml of 1×PBS into 10 L of static waterfor a period of 180 mM. After either 30 or 180 min, the flowing water ineach tank was restored to a final volume of 30 L of water/tank. Fourweeks following a single immersion immunization with the ΔiglC mutant orPBS (control tanks), tilapia fingerlings were challenged by immersion asdescribed above. Briefly, water volumes in each tank were adjusted to 10L of water/tank, and 100 ml of PBS containing F. asiatica suspension wasadded to each tank for a final concentration of 10⁸ CFU/ml of WT F.asiatica. The fish colonization and infection was allowed to progressfor 180 minutes in static water with oxygenation, after which flowingwater was re-assumed to a final volume of 30 L/tank. Three tanks pertreatment were utilized for monitoring mortality every 12 h for 30 days.The remaining tanks were utilized for mucus and serum collection andanalysis. The protective index was calculated according to the formulaas described by Aned 1981: RPS=100%(1−(% mortality in vaccinated fish/%mortality in control fish).

Mucus and serum collection. Mucus and serum collection was performedfollowing as described by Grabowski et al. 2004. Mucus was sampled from5 tilapia fingerlings from each immunized or mocked-immunized group at0, 2, 4, 6, 8 and 10 weeks post-vaccination. Fish were euthanized withan overdose of MS-222 and mucus collected by swabbing both sides of thefish 10 times from head to tail with a cotton applicator. Swabs wereplaced in 1.5 ml microcentrifuge tubes containing 0.9 mL of PBSsupplemented with 0.02% (w/v) sodium azide. Tubes were stored overnightat 4° C. The next morning, tubes and swabs were vigorously vortexed for2 min and liquid removed from the swabs by pressing against the side ofthe tube. The resulting liquid was centrifuged at 3000×g for 10 min andthe supernatant collected and frozen at −20° C. in polypropylene tubesuntil analyzed by ELISA.

For serum analyses, blood samples were obtained from the same fish ateach time point by caudal venepuncture and collection with plainFisherbrand micro-hematocrit capillary tube (Fisher Scientific,Pittsburgh, Pa.). Blood was held at 25° C. for 1 h, and then the serumseparated from cells with centrifugation at 400×g for 5 min and thenstored at −20° C. for later analysis by ELISA.

ELISA. An ELISA was developed to quantify anti-F. asiatica antibodyproduced by tilapia in serum and mucus. Immulon II 96-well flat-bottommicrotitre plates (Thermo Labsystems, Franklin, Mass., USA) were coatedovernight at 4° C. with a 7 ug protein mL⁻¹ solution of sonicated F.asiatica whole cell antigen in 0.05 M carbonate coating buffer, pH 9.6,at 100 μL per well. Plates were then washed three times in PBScontaining 0.05% Tween-20 (PBST). The wells were blocked for 1 h at roomtemperature (RT) with filter-sterilized PBS containing 0.05% Tween 20(Sigma) and 1% bovine serum albumin (BSA; Fraction V, Sigma) (PBST-BSA).Tilapia serum and mucus samples were diluted 1:1000 or 1:50(respectively) in PBST-BSA, and 100 μL of the resulting solution wasadded to three replicate wells of the microtitre plate. The plate wasincubated at 25° C. for 2 h and washed 3× with PBST. Mouse anti-tilapiaIgM heavy chain specific monoclonal antibody (USDA/ARS, Auburn, Ala.)was diluted 1:100 in PBST and 100 μL of this solution added to each wellas described by Shelby et al. 2002. The plate was incubated at 25° C.for 1 h and washed 3× with PBST. Peroxidase-conjugated goat anti-mouseIgG (Pierce Biotechnology, Rockford, Ill., USA) was diluted 1:10,000 inPBST and added to each well. After incubation at 25° C. for 1 h, theplate was washed again 3× in PBST and 100 μL of ABTS PeroxidaseSubstrate System (KPL, Gaithersburg, Md.) was added to each well. TheELISA reaction was stopped after 30 min with 100 μL 1% sodium dodecylsulfate (SDS), and the optical density (OD) of the reactions was read at405 nm with a SpectraMax M2/M2e Microplate Readers (Molecular Devices,Sunnyvale, Calif.). The relative amount of specific antibody wasmeasured as the OD value.

Adoptive transfer studies. Twenty adult fish serum donors were immunizedby intraperitoneal (IP) vaccination with the F. asiatica ΔiglC mutant.Prior to challenge the fish were anesthetized with MS-222 (100 mg/l).Intraperitoneal vaccinated fish received a 0.1 ml injection of bacterialsuspension (10⁷ CFU/fish). Serum collected from the 20 immunized fish at4, 5, and 6 weeks post-immunizations were pooled together and wasanalyzed by ELISA. Endpoint titers were reported as the reciprocal ofthe last dilution yielding an OD more than twice that of the serum fromnaïve control fish. Normal fish serum was obtained from 20 adult naïvefish injected with 1×PBS and processed the same way as the immunizedfish. Heat-inactivated immunized serum (HIIS) and heat-inactivatednormal serum (HINS) were obtained by incubating serum obtained fromimmunized and mocked immunized fish at 56° C. for 30 min. Two hundredand forty naïve tilapia fingerlings (20/tank) were injected IP witheither 200 μl of pooled HIIS, HINS or PBS 24 h before IP challenge withF. asiatica WT. Three tanks per treatment were challenged with either10³, 10⁴, 10⁵, or 10⁶ CFU F. asiatica/fish by IP injection. During thesubsequent 21-d challenge period, fish were monitored daily for clinicalsigns of disease and mortality. Moribund and dead fish were removedtwice daily, and bacterial samples were aseptically obtained from thespleen of morbid and dead fish to confirm the presence of F. asiatica.The LD₅₀ for each treatment was calculated by the method of Reed-Muench(1938), at day 21 post-injection.

Direct Complement Lysis. F. asiatica WT and E. coli DH5a were culturedas described above. Bacteria were adjusted to a concentration of 1×10⁷CFU/ml in PBS. A 1:1 ratio of the bacterial isolates to either PBS,normal (NS) or immunized (IS) tilapia serum was combined, and thesamples were incubated for 2 h at room temperature. Following incubationsub-samples of the bacteria/serum mixtures were collected at 0, 1, and 2h, serially diluted in PBS, and spotted onto either CHAH (F. asiatica)or LB (E. coli) plates.

Opsonophagocytosis assays. To examine the opsonic potential of theimmune sera, an opsonophagocytosis assay was established. Tilapiahead-kidney derived macrophages (HKDM) were collected and purified asdescribed in Neumann et al. 1998 and Secombes 1992. To infect tilapiaHKDM, 5 day cultures of tilapia HKDM in 96 well plates containing1−5×10⁵ cells/well were used. F. asiatica was grown for a period of 8 hin MMH at 25° C. Optical density (OD₆₀₀) of the culture was determined,and the bacteria were adjusted to a final concentration of 5×10⁸ CFU/ml.One ml aliquots of the bacterial suspension was pelleted at 10,000×g for5 minutes in an Eppendorf 5415 D centrifuge (Eppendorf-Brinkman,Westbury, N.Y.), and the pellet was resuspended in either 1 ml of HINSor HIIS. Ten-fold serial dilutions were plated on CHAH after incubationto determine total bacterial cell viability. After 1 h incubation, the96 well plate was inoculated with 10 μl of opsonized bacteria per wellto achieve a multiplicity of infection (MOI) of 50 bacteria: 1macrophage. The plates were centrifuged for 5 min at 400×g tosynchronize bacterial contact with macrophages. Following 2 h incubationat 25° C. with 5% CO₂, the cells were washed three times with warm media(25° C.), further incubated with fresh media and lysed for 15 min attime 0, 24, and 48 h by the addition of 100 of 1% Saponin in PBS. Thelysates were serially diluted and spread onto CHAH plates to determineviable counts. Experiments were performed in triplicate on a minimum ofthree separate occasions with similar results.

Statistical Analysis. The Statistical Analysis System (SAS Institute,Inc. 2003) was used with the general linear models procedure (PROC GLM)to conduct analysis of variance (ANOVA) of a factorial arrangement oftreatments. When the overall test indicated significance, pairwisecomparisons of main effects were calculated with Tukey's test.Interaction effects were examined with pairwise t-test comparison ofleast-square means. For the mortality studies the percent mortalitieswere transformed with an arcsine transformation to normalize the data.To ensure overall protection level of Type I error, only probabilitiesassociated with pre-planned comparisons were used. All comparisons wereconsidered significant at P<0.05.

EXAMPLE 9 Attenuated F. asiatica ΔiglC Mutant Induced ProtectiveImmunity Part 2—Immersion Vaccination with ΔiglC Protected TilapiaFingerlings Against Homologous F. asiatica Immersion Challenge

To evaluate the efficacy of the F. asiatica ΔiglC mutant in protectingtilapia fingerlings against virulent F. asiatica immersion challenge,tilapia fingerlings were vaccinated by immersion by four differenttreatments. Vaccination with a dose of 10⁷ CFU/ml of water for a periodof 30 or 180 min conferred 68.75% and 87.5% relative percent survival(RPS) respectively, against otherwise lethal (80% mortality) immersionchallenge with the wild-type (WT) isolate during a period of 30 days.FIG. 6 shows the mean percent survival of tilapia vaccinated withdifferent treatments of F. asiatica ΔiglC mutant by immersion, or mockvaccinated with PBS (Controls) and challenged 4 weeks later with WT F.asiatica. Fish were vaccinated with: A. 10⁷ CFU/ml of the ΔiglC mutantfor 180 min. B. 10⁷ CFU/ml of the ΔiglC mutant for 30 min. C. 10³ CFU/mlof the ΔiglC mutant for 180 min. D. 10³ CFU/ml of the ΔiglC mutant for30 min. E. PBS for 180 min. Four weeks post-immunization fish werechallenged with 10⁸ CFU/ml of WT F. asiatica for 180 min. Mean percentsurvival for FIG. 6 was calculated 30 days post-challenge with WT. Eachbar represents the mean percent survival±standard error of three tanks(15 fish/tank). An “*” Denotes significant differences, P<0.05 withrespect to the control group by a Student's t-test.

As shown in FIG. 6, vaccination with a dose of 10³ CFU/ml of water for aperiod of 30 min or 180 min conferred 56.25% and 62.5 RPS respectively,against immersion challenge with the WT isolate. Mock (PBS)-vaccinatedfish succumbed to the infection by day 7, presenting clinical signs ofthe disease, including ascites and widespread granulomas in spleen andkidney. Fish vaccinated with either treatments of ΔiglC mutant hadsignificantly higher survival rates than those mocked vaccinated withPBS after challenge with WT F. asiatica (p<0.05) (FIG. 6).

FIG. 7 shows the serum anti-F. asiatica antibody response in activelyimmunized tilapia fingerlings. Fish were vaccinated with on of thefollowing: A. 10⁷ CFU/ml of the ΔiglC mutant for 180 min. B. 10⁷ CFU/mlof the ΔiglC mutant for 30 min. C. 10³ CFU/ml of the ΔiglC mutant for180 min. D. 10³ CFU/ml of the ΔiglC mutant for 30 min. E. PBS for 180min. At four weeks post-immunization, fish were challenged with 10⁸CFU/ml of WT F. asiatica for 180 min. Antibodies were measured during 10weeks post-vaccination (every 2 weeks) as described above. Serum wasdiluted 1:1000. Mean OD values were calculated for each treatment everytwo weeks. Each point represents the mean OD value±standard error of 5fish samples (serum). * Denotes significant differences, P<0.05 withrespect to the control group by a Student's t-test.

FIG. 8 shows the mucus anti-F. asiatica antibody response in activelyimmunized tilapia fingerlings. Fish were vaccinated with: A. 10⁷ CFU/mlof the ΔiglC mutant for 180 min. B. 10⁷ CFU/ml of the ΔiglC mutant for30 min. C. 10³ CFU/ml of the ΔiglC mutant for 180 min. D. 10³ CFU/ml ofthe ΔiglC mutant for 30 min. E. PBS for 180 min. Four weekspost-immunization fish were challenged with 10⁸ CFU/ml of WT F. asiaticafor 180 min. Antibodies were measured during 10 weeks post-vaccination(every 2 weeks) as described above. Mucus was diluted 1:50. Mean ODvalues were calculated for each treatment every two weeks. Each pointrepresents the mean OD value±standard error of 5 fish samples (mucus).*Denotes significant differences, P<0.05 with respect to the controlgroup by a Student's t-test.

As shown in FIGS. 7 and 8, juvenile tilapia vaccinated with eithertreatment of ΔiglC mutant generated a weak serum and mucosal antibodyresponse that wasn't significantly different than that of controls at 2,4 and 6 weeks post-vaccination. However, after the WT immersionchallenge, the serum and mucosal samples from ΔiglC mutant vaccinatedfish with a dose of 10⁷ CFU/ml of water for a period of 30 and 180 min,resulted in a significantly greater secondary antibody response at week8 and 10 post-initial vaccination (p<0.05) (FIG. 7, FIG. 8). Thenon-immunized fish showed an increased primary antibody response afterWT challenge when compared to antibodies levels at week 0 (FIGS. 7 and8).

EXAMPLE 10 Attenuated F. asiatica ΔIglC Mutant Induced ProtectiveImmunity Part 2—Antibodies Partially Contribute to the ProtectionConferred by Vaccination with the F. asiatica ΔIglC Mutant

Intraperitoneal injection with the F. asiatica ΔiglC mutant induced astrong humoral response in adult tilapia and enhanced the production ofantibodies. The pooled immunized sera presented antibody titers >52,000.To test the functional ability of such antibodies, opsonophagocitic andkilling assays were performed, as well as passive immunization trials.

F. asiatica susceptibility to direct effects of IS was compared withthat of E. coli, after mixing the bacteria strains with PBS, normalserum (NS) or immunized serum (IS) for a period of 2 h. Both IS and NScompletely inhibited growth of the E. coli isolate. In contrast, neitherIS or NS had an effect on the growth of F. asiatica in vitro (data notshown).

To test the functional ability of antibodies against F. asiatica in theheat-inactivated immunized serum (HIIS) to mediate phagocytic uptake ofF. asiatica WT, a complement-independent opsonophagocytic assay usingtilapia head kidney derived macrophages (HKDM) was utilized. FIG. 9shows the enhanced antibody-dependent phagocytosis of F. asiatica byHKDM. F. asiatica was opsonized with heat-inactivated immunized (HIIS)or heat-inactivated normal (HINS) sera obtained from adult tilapia.Phagocytosis assays were performed with tilapia HKDM (MOI 1:50) asdescribed above. Results are shown as mean Log₁₀, CFU/ml of F. asiaticauptake in HKDM at 0, 24, and 48 h time point. The error bars representstandard error of triplicate samples, and the results shown arerepresentative of three independent experiments. Different lettersdenote significant differences between treatments, P<0.05. (FIG. 9).Heat-inactivated sera prepared from tilapia immunized with the ΔiglCmutant efficiently mediated phagocytosis of the WT F. asiatica, whereasHINS opsonophagocytosis ability was significantly lower (p<0.05).Bacteria taken up by the HKDM efficiently grew regardless of beingopsonized or not with antibodies (FIG. 9).

Due to the strong antibody response observed in immunized fish, a seriesof passive transfer experiments were performed to determine whetherthese antibodies could prevent infection in vivo. Naive tilapiafingerlings received IP injections of PBS, HINS or HIIS sera (200 μL)collected from adult tilapia immunized with 10⁷ CFU/fish. The tilapiafingerlings were then challenged (IP) with either 10³, 10⁴, 10⁵ or 10⁶CFU/fish of WT F. asiatica and were monitored daily for health andsurvival for a total of 21 days post challenge. FIG. 10 shows theadoptive transfer of heat-inactivated normal serum (HINS),heat-inactivated immunized serum (HIIS) or PBS to naïve tilapiafingerlings. Immune sera was collected from 20 adult tilapia vaccinatedby intra-peritoneal injection (IP) with the ΔiglC mutant 4, 5 and 6weeks post-vaccination. Sera were pooled, and antibodies titers weremeasured before passively immunized the fingerlings. Normal sera werecollected and pooled form 20 adult tilapia injected with PBS 4, 5 and 6weeks post-injection. Naïve fingerlings (60 fish/treatment) wereinjected IP with 200 μl of pooled HINS, HIIS or PBS 24 h before IPchallenge with 10³, 10⁴, 10⁵ or 10⁶ CFU/fish of F. asiatica WT. Animalswere monitored daily for morbidity and mortality. Results arerepresentative of two independent experiments. Mean percent mortalityfor each treatment was calculated 21 days post-challenge with WT. Eachbar represents the mean percent mortality±standard error of three tanks(20 fish/tank). *Denotes significant differences, P<0.05 with respect tothe control group (PBS) by a Student's t-test.

Although passive immunization of HIIS did not protect'against high dosesof the bacterium (10⁶ CFU/fish) injected in the peritoneum of naïvefingerlings, significant (p<0.05) reductions in mortality were observedin HIIS immunized fish when challenged to 10⁴ and 10⁵ CFU/fish andcompared to those immunized with PBS or HINS (FIG. 10).

A live attenuated vaccine given to the fish by the immersion route hasthe advantage of directly targeting the natural routes of attachment andpenetration of the bacteria into the fish and hence inducing protectiveimmunity at the primary site of infection. Results showed that animmersion vaccination with four different treatments of a ΔiglC mutantsignificantly (p<0.05) protected tilapia fingerlings against homologousF. asiatica immersion challenge (FIG. 6). Results of immunization trialsindicated that when the ΔiglC mutant vaccine was delivered for either 30or 180 m at a dose of 10⁷ CFU/ml, relative percent survival (RPS) valuesof 68.75% and 87.5% were obtained, demonstrating the potential of thevaccine to prevent francisellosis in tilapia. During the first 4 weekspost-vaccination, a relatively small antibody response was observed inimmunized fish, and they weren't significantly different to thoseobserved in the control groups. However, upon exposure to WT F.asiatica, a significantly higher (p<0.05) mucosal and humoral antibodyresponse was evident in the fish vaccinated with a dose of 10⁷ CFU/ml(FIG. 7, FIG. 8).

The passive immunity studies described here demonstrate that F.asiatica-specific antibodies mediate protection after IP injection ofdifferent concentration of F. asiatica WT (FIG. 10). Thus the F.asiatica-specific antibody response is a useful component of theprotective immune response to lethal F. asiatica infection in fish.Since F. asiatica is a facultative intracellular organism, the bacteriacan exist in an extracellular form in the tilapia, and thus theantibodies may be able to prevent the systemic spread of bacteria.

An attenuated strain of F. asiatica (ΔiglC mutant) was discovered as alive-vaccine to protect fish from francisellosis, especially F.asiatica. Immunization of tilapia nilotica with the ΔiglC mutant byimmersion delivery provided long lasting protective immune responses(p<0.05), as demonstrated by antibodies levels, and the antibodiesdirected to F. asiatica were protective as shown in passive immunitytrials. Without wishing to be bound by this theory, based on thehomology among Francisella spp. of the iglC gene, we believe that thisattenuated strain of F. asiatica (ΔiglC) could be used to provoke animmune response in fish other than tilapia to protect from infection byF. asiatica, for example, tilapia hybrids, hybrid striped bass and threeline grunt. In addition, based on the similarity of the fish Francisellaspp. in general, we believe that vaccination with the F. asiatica ΔiglCmutant could provide at least some immunity against infection from anyfish Francisella pathogen, e.g., F. noatunensis.

EXAMPLE 11 Live-Attenuated F. asiatica as Vectors of HeterologousAntigens

In addition, this F. asiatica ΔiglC mutant may be used not only tovaccinate fish against Francisella, but also to serve as a vector topresent antigens from other pathogens to the fish immune system,therefore serving as vaccines against other known pathogens of fish aswell. Because attenuated F. asiatica retains its invasive properties andcan be administered by immersion, this attenuated strain is an idealcandidate to use as a vector for delivering heterologous antigens forvaccination. The genetic manipulation techniques have been establishedfor attenuated Salmonella strains. The same general techniques will beused here. A number of different genes from viruses, bacteria andparasites have been successfully expressed in attenuated Salmonella andthe recombinant strains used to immunize small animals. See review inRoberts et al. (1994), and Kang et al. 2002.

Briefly the same techniques as described above will be used to createiglC mutations where the inserted sequences contain both the kanamycinresistance gene to facilitate selection (or another selection marker)and also a gene encoding the heterologous antigen. Preferably the genefor the heterologous antigen is placed under the control of the nativepromoter for the iglC gene or the promoter for the selection marker toensure the antigen is expressed and is seen by the fish immune systembefore it is cleared.

These vaccines are preferably administered to relatively young fishraised in a specific pathogen free environment so the fish will have nopre-existing immunity to the wild type of the carrier strain. Suchpre-existing immunity could cause the carrier strain to be cleared tooquickly.

Heterologous antigens would be selected from those found in otherimportant tilapia pathogens, Streptococcus agalactiae and S. iniae.Examples of heterologous antigens from S. agalactiae would be thesurface immunogenic protein sipA, the cell surface associated proteincspA, and the components of the general protein secretion pathway secY.The antigens from S. iniae would be the hemolysins and M proteins.Currently there are no important virus diseases of cultured tilapia.

EXAMPLE 12 Assay for Specific Identification of Francisella asiatica

Bacterial Strains. The bacterial strains used in this project werechosen because they represent common bacterial fish pathogens, or aremembers of the genus Francisella. Strain F. asiatica LADL 07-285A,isolated from diseased cultured tilapia (Oreochromis spp.) was chosen asa representative of the warm water strain of fish pathogenic F.asiatica. The majority of the isolates tested were recovered by theLouisiana State University, School of Veterinary Medicine (LSU-SVM),Louisiana Aquatic Diagnostic Laboratory (LADL), from diseased fish,while others were acquired from the American Type Culture Collection(ATCC). Francisella tularensis subsp. novicida U112 and F. tularensissubsp. holarctica (LVS isolate) DNA were obtained from the Department ofBiology, University of Texas, San Antonio, Tex. Francisella noatunensissubsp. noatunensis is a recently described member of the genusFrancisella isolated from farmed Atlantic cod (Mikalsen et al. 2007;Ottem et al. 2009; Mikalsen & Colquhoun 2009), and was obtained fromNational Veterinary Institute, Bergen, Norway. Francisella isolates #1,#2, and #3 recovered from moribund hybrid striped bass (Ostland et al.2006) and F. victoria recovered from tilapia (Kay et al. 2006) andshowing >99% identity with F. noatunensis subsp. orientalis after 16SrDNA sequence comparison, were obtained from the University ofWashington, Seattle, Wash. Francisella asiatica LADL 07-285A was grownin cystine heart agar with hemoglobin (CHAH) supplemented as outlined inSoto et al. 2009a, for 48 h at 28° C. Francisella noatunensis subsp.noatunensis was grown in a similar manner but was incubated at 20° C.for 5 d. Flavobacterium columnare was grown on dilute Mueller Hintonagar for 48 h at 28° C. Mycobacterium marinum and Nocardia seriolae weregrown on Lowenstein Jensen slants for one week at 28° C. All the otherbacteria used in the study were grown on blood agar (BA) 5% sheep bloodplates for 48 h at 28° C.

Template DNA Preparation. Bacterial cultures grown on agar media weresuspended in 1 ml of 1× phosphate buffered saline (PBS) and 200 μl wasused for nucleic acid isolation following the manufacturer's protocol inthe High Pure PCR Template Preparation Kit (Roche Diagnostics, Mannheim,Germany). Nucleic acid was also extracted from a negative controlconsisting of 1× sterile PBS alongside of the unknowns to ensure nocross-contamination occurred during the extractions.

TagMan Primers and Probe. The TaqMan primers and probe used in thisstudy were designed based on the nucleotide sequence comparison of theiglC gene of F. tularensis subsp. novicida U112 iglC (GeneBank Accessionnumber AY293579), F. tularensis subsp. holarctica FTNF002-00 (GeneBankAccession number CP000803) iglC, F. tularensis subsp. mediasiatica FSC147 iglC (GeneBank Accession number CP000915), F. philomiragia ATCC25017 iglC (GeneBank Accession number CP000937), and F. noatunensissubsp. orientalis LADL 07-285A iglC (GeneBank Accession number FJ386388)(Table 3). The primers and probe were designed following the real-timeqPCR Assay Design Software (Biosearch Technologies, San Francisco,Calif., USA). Primers and probe concentration were optimized at thebeginning to determine the minimum primer concentrations giving themaximum ΔRn, and the minimum probe concentration that gave the minimumC_(T). The optimization was done according to the TaqMan Universal PCRMaster Mix manufacturer (Applied Biosystems, Foster City, Calif., USA).

TABLE 3 TaqMan primers and probe used in this study PrimersMelting temperature and Probes 5'-3' Sequence (C°) iglC forwardGggcgtatctaaggatggtatgag 66.36 (SEQ ID NO: 21) iglC reverseAgcacagcatacaggcaagcta 66.63 (SEQ ID NO: 22) iglC probeFAM atctattgatgggctcacaacttcacaa BHQ-1 68.34 (SEQ ID NO: 23)

Real-time TagMan PCR Assays. The real-time PCR assays were conducted andanalyzed within the Applied Biosystems 7500 Fast Real-Time PCR Systems(Applied Biosystems). The 25 μl reaction mixture consisted of a TaqManUniversal PCR Master Mix (Applied Biosystems), containing 10 μmol ofeach primer, 3 μmol of probe and 5 μl of DNA extracted sample. Templatecontrols containing PCR grade water and seven serial dilutions of 100 ngμl⁻¹ of F. asiatica isolate LADL 07-285A diluted in PCR grade water andmeasured in a NanoDrop Spectrophotometer ND-V3.5 (Nanoprop TechnologiesInc., Wilmington, Del.) were included in each run. The unknown samples,as well as the diluted standards and negative controls were run intriplicate. Cycling conditions were 2 min at 50° C., 15 min at 95° C.followed by 40 cycles of 15 s at 95° C., 60 s at 60° C.

The assay was found to be specific for the warm water fish pathogen, F.asiatica and no evidence of crossreactivity was detected (no significantelevated signal was observed with any of the other tested bacterial DNA)(Data not shown).

Sensitivity of the Real Time PCR Assays. For sensitivity determination,the TaqMan assays were evaluated by two different independent methods.Three separate extractions of F. asiatica DNA were adjusted to aconcentration of 100 ng μl⁻¹ Nanoprop Spectrophotometer ND-1000 V3.5(Nanodrop Technologies Inc., USA), and ten fold dilutions were made inPCR grade water until reaching a concentration of 1 fg μl⁻¹. Genomeequivalent (GE) calculation was based on assuming a 2-MB genome size forF. philomiragia and several subspecies of F. tularensis. Fordetermination of colony forming units (CFU), several isolated coloniesof F. asiatica were picked from a fresh CHAH culture and suspended in 1ml of phosphate buffered saline (PBS) pH 7.2, until an OD₆₀₀ of 0.75 wasreached and measured in a DU-640 Spectrophotometer (Beckman CoulterInc., Brea, Calif., USA). Ten fold serial dilutions in PBS were madefrom this sample, and colony counts were performed on CHAH by the dropplate method to verify bacterial numbers. Extraction of DNA from 200 μlof each dilution was used for CFU quantification in the real time PCRassay. Amplification efficiencies were determined and all assays wererun in triplicate.

Sensitivity of the Real Time PCR Assay in Fish Spleen. In order todetermine the sensitivity limit of the assay, triplicate samples of onegram of uninfected tilapia spleen (recently acquired fresh tissue) werehomogenized with a Kontes PELLET PESTLE® Micro Grinder (A. Daigger andCompany Inc., 620 Lakeview Parkway, Vernon Hills, Ill., USA) in a 4 mlsuspension of early stationary phase F. asiatica cells diluted in PBS toa final concentration of 2, 20, 200, 2×10³, 2×10⁴, 2×10⁵, 2×10⁶, 2×10⁷CFU g tissue⁻¹. Two hundred microliters of the homogenates containingapproximately 50 mg of spleen, were centrifuged at 12 000 g for 1 minand DNA extracted following the manufacturers protocol “Isolation ofNucleic Acids from Mammalian Tissue”, High Pure PCR Template PreparationKit (Roche Diagnostics, Mannheim, Germany): Enumeration of F. asiaticaby real-time PCR was compared with plate count values, taking intoaccount dilution/concentration factors due to volumes used in DNAextraction and final elution volumes. Amplification efficiencies weredetermined and all assays were run in triplicate.

The sensitivity of the assay was determined using a triplicate dilutionseries from 0.5 fg reaction⁻¹ to 1.4 mg reaction⁻¹ of F. asiaticagenomic DNA. The lowest amount of detection was determined to be 50 fgof DNA (equivalent to ˜25 GE). Threshold cycle (Ct) determined by TaqManreal-time PCR amplification of DNA, extracted from serial dilutions ofpure F. asiatica bacterial culture, showed a linear (R²=0.994)relationship with log numbers of CFU from 2.5×10⁷ to 2.5×10¹ CFU ml⁻¹based on plate counts (Data not shown). Ten fold serial dilutions ofnucleic acid extracted from the initial dilutions of the pure bacterialculture also showed a linear relationship between the log amount ofnucleic acid and the TaqMan real-time PCR Ct from 1.4 mg to 50 fg.Linear detection of amplified product was also revealed in seriallydiluted F. asiatica spiked spleen homogenates (R²=0.985) (Data notshown). This indicates that the presence of tissue homogenate did notimpede the sensitivity of the real-time PCR assay within this range ofCFUs. Uninfected tilapia spleen and water controls showed no signalafter 40 cycles.

Experimental Infectivity Trial. The tilapia fingerlings used during thetrial were obtained from a source with no history of Francisellainfection and a sub-sample of the population was confirmed as negativefor Francisella bacteria by complete clinical, bacteriological andmolecular analysis as described in Soto et al. 2009a. Fish weremaintained at 10 fish per tank and fed commercial tilapia feed daily(Burris Aquaculture Feeds, Franklinton, La.) at ˜3% fish body weight perday. The mean weight of the fish was 9.1 g and the mean length was 18cm. Three tanks were used per treatment, and one tank was used as acontrol. Fish were immersed in 8 L of static water containingapproximately 3.7×10⁷ CFU/ml in tank water for 3 h at 23-25° C., andthen the volume of the tanks was adjusted to 20 liters with cleanoxygenated water. Control fish were treated in a similar manner, butreceived sterile PBS.

Following each challenge exposure, mortality was recorded every 12 h for30 d. Prior to collection of spleen, moribund and survivor fish wereeuthanized with an overdose of MS-222. The spleens from dead, moribundand survivor fish were collected aseptically in 1.5 microcentrifugetubes (Fisherbrand, Fisher Scientific, USA), weighed, and DNA wasextracted from ˜20 mg of spleen following the manufacturers protocol“Isolation of Nucleic Acids from Mammalian Tissue”, High Pure PCRTemplate Preparation Kit (Roche Diagnostics, Mannheim, Germany). Therest of the tissue was homogenized in ˜50 μl PBS and plated on CHAH. Theeluted DNA was stored at 4° C. until used.

At 30 days following challenge, the mean mortality in the tanks was56.6%. In order to test the ability of the iglC TaqMan assays toidentify F. asiatica in tilapia tissue, spleens from infected fish wereanalyzed. One hundred percent of the morbid and survivor (challenged)fish were positive by the assay, and all non-challenged fish werenegative. Detection of the bacteria by culture on CHAH agar media waspossible in 58% of the dead fish, and in 38% of the survivors. The meanamount of F. asiatica GE detected in spleens from dead fish analyzed byreal time PCR was 1.8×10⁵ GE mg⁻¹ of spleen tissue, while surviving fishpresented a mean amount of 1.5×10³ GE mg⁻¹ of spleen tissue.

A TagMan real-time quantitative PCR assay for the rapid identificationand quantification of the emergent fish pathogen F. asiatica was thusdeveloped. The development of this highly sensitive diagnostic methodwill enhance the diagnosis of this fastidious organism that could bepresent at low levels in fish tissue. The assay developed in this studyis directed against the previously identified iglC gene in F. asiaticaisolate LADL 07-285A. The specificity of the TaqMan probe real-time iglCPCR assay was assessed with other strains of the genus Francisella (F.tularensis subsp. novicida U112 and F. tularensis subsp. holarcticaLVS), clinically relevant cold and warm water fish pathogens (F.noatunensis subsp. noatunensis, Streptococcus spp., Edwardsiella spp.,Aeromonas spp., Vibrio spp., Mycobacterium spp., Photobacterium spp.,etc), and non-infected tilapia splenic tissue. After 40 cycles, DNAsamples from these strains failed to show amplification using the realtime PCR assay, and the assay showed no cross reaction of the chosenprimers and probe with fish tissue or opportunistic fish pathogenslisted above. This is particularly important with francisellosis sincemoribund and dead fish are commonly found with secondary infections, andattempts to isolate Francisella spp. can be very difficult due to thefastidious nature of the organism. The high specificity achieved by theTaqMan real time PCR assay did not amplify the closely related coldwater pathogen F. noatunensis subsp. noatunensis, but it did amplifyrepresentative F. asiatica isolates recovered from warm water culturedtilapia and striped bass.

The sensitivity limit of the assay was found to be ˜50 fg of DNA(equivalent to ˜25 GE or CFU) of F. asiatica. Different approaches wereused to verify that our DNA extraction methodology and the real time PCRassay did not interfere with the results obtained in the assay. Aftersuspending viable live bacteria in tilapia tissue homogenates and inPBS, performing CFU counts in CHAH, extracting the DNA under the sameconditions, and running the assay, it was found that fish tissue did notnegatively affect the real-time PCR detection or quantification of F.asiatica.

When experimentally infected, tilapia were used to simulate wildepizootics, the real-time PCR assay enabled detection of the bacteriumin all the dead, moribund and surviving fish 30 days post challenge,whereas it was possible to isolate the bacteria by conventionalculturing on agar plates in only 58.8% (10 of 17) of dead and moribundfish, and in 38% (5 of 13) of the survivor fish after 30 days postchallenge. The real-time PCR assay was also able to detect the presenceof F. asiatica in the water of infected fish by filtering the water andextracting DNA from the filter for use in the real-time PCR assay.

Thus an iglC based TaqMan real-time PCR assay was developed with highsensitivity and specificity for the detection and quantification of theemergent warm water fish pathogen F. asiatica. The assay can be used notonly as a rapid diagnostic test for francisellosis, but can also be usedas a research tool for bacterial persistence, drug therapy efficacy,epidemiological studies, screening of broodstock fish, and detection ofreservoirs for infection.

As used in the Claims, “tilapia” is used as a generic term to designatefish members of the three known genera of tilapia, Tilapia,Sarotherodon, and Oreochromis, including hybrids among the species. Forexample, the term would encompass the Nile tilapia (Oreochromisniloticus), and the hybrid red tilapia, Oreochromis mossambicus x O.niloticus.

As used in the Claims, a “protective amount” of an attenuated bacteriumis an amount that, when administered to a fish as a vaccine, induces adegree of immunity sufficient to reduce to a statistically significantdegree the susceptibility of the fish to an infection by a pathogen, inthis case, to species of the genus Francisella.

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The complete disclosures of all references cited in this specificationare hereby incorporated by reference. Also incorporated by reference isthe complete disclosure of the following: (1) E. Soto et al.,“Attenuation of the fish pathogen Francisella sp. by mutation of theiglC gene,” Journal of Aquatic Animal Health, vol. 21, pp. 140-149(2009), epub Oct. 12, 2009; (2) E. Soto et al., “Francisella sp., anemerging pathogen of tilapia, Oreochromis niloticus (L.) in Costa Rica,”epub. Jun. 8, 2009, Journal of Fish Disease, vol. 32, pp. 713-722(2009); (3) E. Soto et al., “Interaction of Francsiella asiatica withTilapia (Oreochromis niloticus) innate immunity”, Infection andImmunity, vol. 78, pp. 2070-2078; epub Feb. 16, 2010 (2010); (4) E. Sotoet al., Development of a Real-time PCR assay for identification andquantification of the fish pathogen Francisella noatunensis subsp.Orientalis,” Diseases of Aquatic Organisms, vol. 89(3), pp. 199-207;epub Apr. 9, 2010 (2010); and (5) E. Soto et al., In vitro and in vivoefficacy of florfenicol for treatment of Francisella asiatica infectionin tilapia, Antimicrobial Agents and Chemotherapy, Aug. 16, 2010 (Epubahead of print). In the event of an otherwise irreconcilable conflict,however, the present specification shall control.

1. An attenuated Francisella bacterium found in fish comprising a mutantiglC gene.
 2. The attenuated bacterium as in claim 1, wherein thebacteria is Francisella asiatica.
 3. The attenuated bacterium as inclaim 1, wherein said mutant iglC gene has an insertion mutation of atleast 100 base pairs as compared to the iglC gene of a wild-typeFrancisella asiatica bacterium.
 4. The attenuated bacterium as in claim1, wherein the attenuated bacterium is the attenuated Francisellaasiatica bacterium with ATCC Accession Number PTA-11268.
 5. A vaccinecomprising a protective amount of an attenuated Francisella bacterium asrecited in claim
 1. 6. A vaccine comprising a protective amount of anattenuated Francisella bacterium as recited in claim
 2. 7. A vaccinecomprising a protective amount of an attenuated Francisella bacterium asrecited in claim
 3. 8. A vaccine comprising a protective amount of anattenuated Francisella bacterium as recited in claim
 4. 9. The vaccineas in claim 5, wherein said bacterium additionally comprises anexogenous gene encoding an antigenic peptide or antigenic protein thatis native to a fish pathogen other than Francisella.
 10. The vaccine asin claim 5, wherein said bacterium additionally comprises an exogenousgene encoding a Salmonella protein selected from the group consisting ofsurface immunogenic protein sipA, the cell surface associated proteincspA, and the components of the general protein secretion pathway secY.11. A method of reducing the susceptibility of a fish to francisellosis,comprising administering to the fish a vaccine as recited in claim 5.12. The method as in claim 11, wherein the fish is selected from thegroup consisting of tilapia, cod, three-line grunt, striped bass, hybridstriped bass, and salmon.
 13. The method as in claim 11, wherein thefish is tilapia, striped bass, hybrid striped bass, and three linegrunt.
 14. The method as in claim 11, wherein the fish is tilapia. 15.The method as in claim 11, wherein said administering step comprisesimmersing the fish in said vaccine.
 16. The method as in claim 11,wherein said administering step comprises feeding the fish a foodproduct comprising said vaccine.
 17. The method as in claim 11, whereinsaid administering step comprises injecting the fish with said vaccineintraperitoneally.
 18. A method of reducing the susceptibility of fishselected from the group consisting of tilapia, three-line grunt, stripedbass and hybrid striped bass to Francisella asiatica, comprisingadministering to the fish a vaccine as recited in claim
 6. 19. Themethod as in claim 18, wherein the fish is tilapia.
 20. A method ofreducing the susceptibility of a fish to an infection by a pathogen thatexpresses the antigenic protein or antigenic peptide that is encoded bythe exogenous gene of the bacterium of claim 9, comprising administeringto the fish a vaccine as recited in claim
 9. 21. The method of claim 20,wherein said exogenous gene is one encoding a Salmonella proteinselected from the group consisting of surface immunogenic protein sipA,the cell surface associated protein cspA, and the components of thegeneral protein secretion pathway secY.
 22. A real-time PCR assay kitfor diagnosing an infection of Francisella asiatica, said kit comprisingthe primers of SEQ ID NO:21 and SEQ ID NO:22, and the probe of SEQ IDNO:23.
 23. A method to diagnosis in a fish an infection of Francisellaasiatica, said method comprising extracting DNA from the fish andassaying the DNA using the real-time PCR assay of claim
 22. 24. A methodto diagnosis an infection of Francisella asiatica by analyzing the wateraround a fish population, said method comprising filtering a sample ofthe water, extracting DNA from the filter, and assaying the DNA usingthe real-time PCR assay of claim 23.